Optimization Of Extraction Process Of Ore-derived Fulvic Acid By Hydrogen Peroxide And Its Anti-hepatoma Effect

Objective: The quality of raw coal from different origins of fulvic acid was evaluated,and the extraction process was optimized.At the same time,an infrared spectrum standard curve with good applicability was established as the same source and the same production process control analysis method.To make a systematic pharmacological test on the anti-tumor effect of fulvic acid,and provide theoretical data for further research,in order to carry out anti-tumor clinical application and drug development of fulvic acid.Test methods: 1 The basic properties of raw coal such as moisture,ash and humic acid were determined,and the quality and quality of the five samples were analyzed.2 In vitro inhibition of tumor cell activity,inhibition rate of tumor activity in vitro and IC50 as indicators to determine the anti-tumor effect of fulvic acid in vitro,screening for the best anti-tumor effect of fulvic acid source.3 The BOX-Behn Ken Design method and Design Exper v11.0.5b software were used to design the experiment.The yield of fulvic acid and IC50,which inhibited the activity of cancer cells in vitro,were used as comprehensive indexes to analyze the results and optimize the extraction process.4 A preliminary structural analysis of the infrared spectrum of fulvic acid was carried out by FT-IR spectrometer,and a control analysis method was established..5 LD50 of fulvic acid was determined by acute toxicity test in mice,which could be used as the dosage basis for the next in vivo experiment in mice.6 Through the establishment of H22 tumor-bearing mouse model,the general conditions,tumor inhibition rate and immune organ index of mice were used as indicators to investigate the inhibitory effect of fulvic acid on H22 liver cancer xenografts in mice.7 After confirming the exact anti-tumor effect of fulvic acid,the anti-tumor mechanism of fulvic acid was investigated by flow cytometry from the aspects of inducing apoptosis and improving autoimmune function.Results: 1 The anti-tumor effect of fulvic acid in vitro was preliminarily determined by the inhibition rate and IC50 of fulvic acid in vitro,and the best source was determined as Sample 3.2 The optimum extraction technology of fulvic acid is as follows: liquid-solid ratio 3:1,oxidant concentration 15%,and oxidation time 90%.Under the condition of this parameter,the IC50 of the inhibitory activity of fulvic acid on the two hepatocellular carcinoma cells(BEL-7402,Hep G2)was the minimum.3 The LD50 of fulvic acid was 2.4255 g and the 95% confidence interval of LD50 was(2.0687,2.8439)by acute toxicity test in mice.4 The IC50 of fulvic acid inhibiting Hep G2 activity was 1.5909 mg/ml and that of BEL-7402 activity was 4.4858 mg/ml.Compared with negative control group,the effect of different concentrations of fulvic acid on early and late apoptotic rate of Hep G2 was extremely significant(P < 0.01).5 Compared with the model group,the tumor weight of each group treated with fulvic acid decreased significantly(P < 0.01).Compared with the cisplatin group,the thymus index,spleen index and liver index of the middle dose group of fulvic acid increased significantly(P < 0.01,P < 0.05).6 Compared with the model group,the contents of TNF-a and IL-2 in the fulvic acid groups were significantly increased(P < 0.01).7 Compared with model group,the percentage of CD3 + CD4 + cells in spleen and peripheral blood increased(P < 0.01)and the ratio of CD3 + CD4 + / CD3 + CD8 + increased(P < 0.01,P < 0.05).Conclusion: 1 A fixed source of fulvic acid was identified by preliminary screening of antineoplastic agents in vitro.2 Response surface methodology was used to extract fulvic acid with good repeatability and reliable data.3 Through in vitro experiments,fulvic acid increased the early and late apoptotic rate of cancer cells.4 The anti-tumor mechanism of fulvic acid may be related to the regulation of the ratio of CD3+CD4+/CD3+CD8+ in T lymphocyte subsets.Through the above systematic pharmacological tests,the inhibitory effect of fulvic acid on liver cancer cells was preliminarily confirmed,and data support for the development of anti-tumor of fulvic acid was provided.